Silk fibers have many current and future biomedical applications. They are widely used as fine suture materials and even thinner fibers are needed for ocular, neurological, and cosmetic surgeries. Silk fibers also hold promise as materials for artificial ligaments and tendons and they have many other potential biomedical applications. Many different recombinant protein production systems have been used to try to meet current needs and to develop additional biomedical applications of silks. Each has yielded silk proteins, but none has consistently yielded useful silk fibers. Thus, the overall purpose of this R21 exploratory proposal is to develop a system that can produce spider silk fibers. Our basic approach will be to adopt the silkworm as a surrogate host for spider silk protein production. The highly efficient piggybac system will be used to genetically transform mutant silkworms, which produce no native silk, with a synthetic gene encoding an unusually large spider silk protein. The genetic and biochemical properties of the resulting transgenic silkworms, particularly their ability to produce spider silk fibers, will then be critically assessed. A key feature of our plan is that it includes specific measures that will optimize our ability to obtain fibers. These include: (i) using a synthetic gene encoding an unusually large spider silk protein, (ii) using a promoter that targets expression of the heterologous silk proteins to the silk gland, which is naturally equipped to spin silk fibers, and (iii) using an appropriate leader peptide for secretion from the silk gland, and (iv) using a surrogate host, the silk moth, which is highly amenable to genetic transformation and naturally equipped to spin silk fibers. The successful isolation of a transgenic silkmoth that can produce spider silk fibers in this exploratory project will set the stage for future projects designed to further develop this system to produce spider silk fibers with pre-determined physical properties optimized for specific biomedical applications. This will exploit current knowledge of specific peptide motifs contributing tensile strength or elasticity to spider silks. Theoretically, these motifs can be combined in various ways to design silk fibers differing in strength and elasticity. The overall likelihood that the current project can be successfully completed is enhanced by the fact that it will be undertaken by a team of three researchers with established, complementary programs in three areas key to the project: spider silks (Lewis), insect expression systems (Jarvis), and insect transformation (Fraser). Silk fibers have many current and future biomedical applications. They are widely used as fine suture materials and even thinner fibers are needed for ocular, neurological, and cosmetic surgeries. Silk fibers also hold promise as materials for artificial ligaments and tendons and they have many other potential biomedical applications. Many different recombinant protein production systems have been used to try to meet current needs and to develop additional biomedical applications of silks. Each has yielded silk proteins, but none has consistently yielded useful silk fibers. Thus, the overall purpose of this R21 exploratory proposal is to develop a system that can produce spider silk fibers for these current and future biomedical applications. Our plan for this exploratory project is to isolate a transgenic silkworm that can produce spider silk fibers. If this project is successful, it will set the stage for future projects designed to further develop this system to produce spider silk fibers with pre-determined physical properties optimized for specific biomedical applications. These projects will exploit current knowledge of specific peptide motifs contributing tensile strength or elasticity to spider silks. [unreadable] [unreadable] [unreadable]